Research Goals

Mechanisms to increase protein synthesis are crucial in the development and progression of diverse cancers. While most oncogenic pathways target the translation initiation machinery, the elongation step of translation also represents an important regulatory node. Our work identified METTL13, FAM86A, and SMYD5, previously not recognized protein methyltransferases (PMTs) of ribosomal proteins as critical regulators of tumorigenesis (Nature, 2024; Cell Research, 2024; Molecular Cell, 2024; Cell, 2019).

We found that METTL13 and FAM86A, through methylation of eEF1A and eEF2 respectively, regulate mRNA translation elongation, the most energy-consuming process in the cell, which has a major role in cancer pathogenesis. During translation elongation, the GTPase eEF1A delivers with fidelity aminoacylated tRNAs to translating ribosomes. A peptide bond is made, and eEF2 catalyzes the movement of the peptidyl-tRNA·mRNA complex, allowing the process to repeat. Importantly, there are several methylation sites at eEF1A, eEF2, and other ribosome proteins that are highly conserved, which function is poorly recognized, and in most cases, the specific PMTs generating the modifications remain unknown.

Our investigation of the molecular and cellular consequence of ribosomal protein methylation will provide novel insights into the role of lysine, arginine and histidine methylation in normal and malignant cells and, by analogy to the “histone code,” explore the idea of a ribosomal “methylation code” that calibrates translation outputs.

A central hypothesis to be tested here is that uncovering the function of PMTs that specifically methylate ribosomal proteins may reveal new mechanisms regulating mRNA translation and provide new therapeutic opportunities. We are utilizing the methods we have established to assess the specific consequences of ribosomal protein methylation on global protein synthesis and specific mRNA translation.

Amplifications of chromosomal regions 3q26-29 (3q26^AMP) and 8p11-12 (8p11^AMP) represent two critical genetic hallmarks of LUSC present in >90% of tumors. We have recently identified the H3K36 methyltransferase NSD3 as a principal 8p11^AMP-associated oncogenic driver in LUSC (Nature, 2021).

Our initial studies suggest a mechanism by which 8p11^AMP-associated LUSC is dependent on the NSD3-H3K36me2 axis to sustain transcriptional programs required to promote malignant progression. Our work provided evidence in mouse and human cancer models that NSD3’s catalytic activity regulates LUSC pathogenesis.

Given the co-occurrence of 3q26^AMP and 8p11^AMP, we explore genetic interactions between amplified genes in promoting LUSC malignant progression. Using genetic screening, we found that from over 200 genes within 3q26^AMP, four genes (3q26 onco-cassette) are required and sufficient to trigger the transformation of normal human lung epithelial cells. Based on those findings, we generated a mouse model with conditional overexpression of the identified 3q26^AMP oncogenes (3q26 onco-mouse).

Our research demonstrates that 3q26^AMP and 8p11^AMP oncogenes cooperate in driving an aggressive course of disease progression, similar to what is observed in the majority of LUSC patients with concomitant amplifications. The goal of this research is to decipher the molecular and epigenetic network triggered by 3q26^AMP and 8p11^AMP amplifications on oncogenic programming in LUSC. This research is supported by R01CA278940.

Similarly to NSD3, our work on NSD2 identified recurring gain-of-function mutation (E1099K) and upregulation triggering activation of oncogenic programs to promote KRAS-driven lung adenocarcinoma (LUAD) pathogenesis (Mol Cell, 2022). A striking aspect of NSD-family hyperactivation is not the emergence of a higher number of tumors, but rather the development of rare, exceptionally large, and aggressive tumors, suggesting that the NSD-H3K36me2 axis facilitates tumor evolution. This observation is consistent with the hypothesis that NSD2’s action as an epigenetic mutagen accelerates the ability of tumors to acquire rare yet profoundly tumorigenic, malignant alterations.

We are utilizing cutting-edge epigenetic and single-cell methods to explore the molecular basis for NSD-mediated H3K36me2 synthesis in LUAD and LUSC using novel, clinically relevant models. These experiments will provide, to our knowledge, the first analyses of NSDs’ role in tumor cell lineage diversity/plasticity and ITH.

Finally, with the medicinal chemistry team of Dr. Soth (MD Anderson), Dr. Jian Jin (Mount Sinai) and Dr. Gozani (Stanford), we are developing the first-in-class catalytic inhibitors of NSD2 and 3. Our initial data suggest we are able to achieve high selectivity of NSD2 inhibition at single nM concentrations, and ongoing animal studies indicate favorable PK/PD and low toxicity (Patent: WO2024073282A2).

We are also exploring combination therapies with Kras^G12C inhibitors and immunotherapy, including CAR T cells. This research is supported by R01CA272844, R01CA278940, CPRIT RP220391, foundation grants and sponsored research agreements.

Our work in collaboration with Dr. Gozani (Stanford) identified METTL13, a previously not recognized protein methyltransferase (PMT), as a critical regulator of tumorigenesis. We found that METTL13, through methylation of lysine 55 of eEF1A, regulates mRNA translation, the most energy-consuming process in the cell. Specifically, METTL13-catalyzed eEF1A methylation increased the intrinsic GTPase activity of eEF1A in vitro and protein production in cells. METTL13 deletion and subsequent loss of eEF1A K55me2 did not affect non-transformed cells but greatly attenuated KRAS-driven tumorigenesis in mouse models of pancreatic and lung adenocarcinomas and patient-derived xenografts (PDX) (Cell, 2019).

The ongoing research effort is focused on the role of METTL13 in resistance to Kras^G12C inhibitors via specific METTL13-mediated adaptation of mRNA translation (supported by the R01CA236118). Of note, we have now identified and are currently pursuing research on several enzymes methylating ribosomal proteins and regulating mRNA translation. For instance, in our most recent work we have identified FAM86A as a unique lysine methyltransferase of eEF2 controlling oncogenic ribosomal translation output to lung cancer pathogenesis (Molecular Cell, 2024).

Further, we identified a novel lysine methyltransferase SMYD5 to methylate ribosomal protein rpl40, which regulates mRNA translation elongation and is critically important in regulation in the malignant progression of gastric cancer and hepatocellular carcinoma (Nature, 2024; Cell Research, 2024).

One of our long-term interests is PDAC cells’ capacity to evolve and “re-wire” their signaling networks in response to therapy (e.g., Nature, 2014; Nature Medicine, 2013; Nature Medicine 2015; Genes Dev, 2016). To explore the role of epigenetic factors in the resistance, we performed a genetic screening (in collaboration with Dr. Basik, Stanford) and identified SETD5 putative methyltransferase as a major driver of PDAC resistance to KRAS-MAPK inhibitors.

In collaboration with Dr. Gozani’s lab (Stanford) we found that SETD5 lacks intrinsic enzymatic activity but forms a co-repressor complex (NCOR1/HDAC3/G9a) that selectively deacetylates and methylates histone H3K9, which regulates a network of drug resistance pathways (Cancer Cell, 2020).

We are now testing the preclinical potential of specific inhibition of SETD5 complex activities and its role in resistance to Kras^G12C inhibitors using our novel PDAC models driven by Kras^G12C oncogene in the adult pancreas (supported by R01CA236949). Dr. Mazur is also the scientific founder of Amplify Medicines, Inc. (now part of Ikena Oncology), which seeks to translate the molecular mechanisms of drug resistance into clinical practice to synergistically magnify the efficacy of cancer therapies.

In collaboration with the Bedford Lab (MD Anderson), we performed a proteomic screening (focused on protein methylation). We found that CARM1 arginine methyltransferase specifically methylates transcription pioneering factor NFIB, previously implicated in the malignant progression of SCLC. We found that NFIB methylation by CARM1 is required to trigger genome-wide modification of chromatin accessibility.

Mechanistically, we identified previously unrecognized reader protein TRIM29 that recognizes methylated NFIB and recruits chromatin-modifying enzymes (Nature Communications, 2022). Using several novel animal strains, including those developed in the Mazure Lab, we developed a “two-step” genetic model of SCLC in which initiating mutations can be separated from tumor progression/maintenance, and we have validated CARM1 as a new therapeutic target in SCLC (supported by R01CA272843).

In collaboration with Dr. Reynoird's lab, we identified BCAR3 as a new substrate of SMYD2 using a high-throughput proteomic screening. We demonstrated that cell-adherence regulator BCAR3 methylation is critical for cell motility and invasiveness using advanced molecular and microscopy approaches. We further found through quantitative proteomics a novel methyl-binding domain within actin-regulators FMNLs, able to specifically recognize BCAR3 methylation.

FMNLs are essential in forming protrusive actin structures such as lamellipodia, and our subsequent analysis revealed that methylation of BCAR3 enhances lamellipodia formation by recruiting FMNLs at the cell leading edge, generating force enabling cellular motility critically important for metastatic tumor spread (Cell Discovery, 2024).